WO2001081365A2 - Metal chelating compositions - Google Patents

Metal chelating compositions Download PDF

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Publication number
WO2001081365A2
WO2001081365A2 PCT/US2001/011529 US0111529W WO0181365A2 WO 2001081365 A2 WO2001081365 A2 WO 2001081365A2 US 0111529 W US0111529 W US 0111529W WO 0181365 A2 WO0181365 A2 WO 0181365A2
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cooh
metal chelating
chelating composition
metal
composition
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PCT/US2001/011529
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English (en)
French (fr)
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WO2001081365A3 (en
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William K. Kappel
Venkatappa Viswanatha
Handong Li
Richard J. Mehigh
John G. Dapron
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Sigma-Aldrich Co.
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Application filed by Sigma-Aldrich Co. filed Critical Sigma-Aldrich Co.
Priority to JP2001578453A priority Critical patent/JP4298201B2/ja
Priority to EP01924862A priority patent/EP1276716B2/en
Priority to DE60111628T priority patent/DE60111628T3/de
Priority to AT01924862T priority patent/ATE298321T1/de
Priority to AU2001251479A priority patent/AU2001251479A1/en
Publication of WO2001081365A2 publication Critical patent/WO2001081365A2/en
Publication of WO2001081365A3 publication Critical patent/WO2001081365A3/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J45/00Ion-exchange in which a complex or a chelate is formed; Use of material as complex or chelate forming ion-exchangers; Treatment of material for improving the complex or chelate forming ion-exchange properties
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803General methods of protein analysis not limited to specific proteins or families of proteins
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07BGENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11Compounds covalently bound to a solid support
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/25Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125Digestion or removing interfering materials

Definitions

  • the present invention is generally directed to metal chelating compositions and to methods for making and using the same for protein purification, detection or binding and, in particular, to nitrilotriacetic acid derivatives that have improved binding specificity and stability and to methods for making and using these nitrilotriacetic acid derivatives for protein purification, protein detection or protein binding.
  • nitrilotriacetic acid resin a tetradentate chelator known as nitrilotriacetic acid resin was developed for use with metals having six coordination sites.
  • This resin has become the preferred resin for the purification of poly histidine containing proteins since it has very little metal leaching and good selectivity.
  • considerable amount of effort is required to obtain this selectivity.
  • the addition of various amounts of imidazole is necessary to determine whether the resin will bind the protein selectively and the capacity of the resin for the protein must be optimized to achieve the desired results (Janknecht et all, Proc. Natl . Acad. Sci . , 88:8972-8976, 1991, Schmitt et all., Molecular Biology Reports, 88:223-230, 1993).
  • Dobeli et al describe nitrilotriacetic acid resins suitable for protein purification represented by the general formula:
  • carrier matrix [carrier matrix] -spacer-NH- (CH 2 ) X -CH (COOH) -N (CH 2 COO-) 2 Ni 2+ wherein x is 2, 3 or 4 , the carrier matrix is one used in affinity or gel chromatography such as cross- linked dextrans, agarose or polyacrylamides, and the spacer is preferably -0-CH 2 -CH (OH) -CH 2 - or -O-CO- .
  • Dobeli et al . U.S. Patent No. 4,877,830 at col. 2, lines 23-37.
  • These resins are prepared by reacting an N-terminal protected compound of the formula:
  • R is an amino protecting group and x is 2,3 or 4, with bromoacetic acid in an alkaline medium and subsequently cleaving off the protecting group and reacting this product with an activated resin.
  • compositions and to metal chelates which are relatively stable and provide superior binding specificity for protein or polypeptide purification, protein or polypeptide detection or protein or polypeptide binding, and the provision of processes for the preparation and use of such compositions.
  • the present invention is directed to a metal chelating composition having the formula:
  • Q is a carrier
  • S 1 is a spacer
  • A is an ether, thioether, selenoether, or amide 1inkage ;
  • T is a bond or substituted or unsubstituted alkyl or alkenyl ;
  • X is -(CH 2 ) k CH 3 , - (CH 2 ) k COOH,- (CH 2 ) k S0 3 H, - (CH 2 ) k P0 3 H 2 , - (CH 2 ) k N(J) 2 , or - (CH 2 ) k P(J) 2 ;
  • k is an integer from 0 to 2 ;
  • J is hydrocarbyl or substituted hydrocarbyl
  • Y is -COOH, -H, -S0 3 H, -P0 3 H 2 , -N(J) 2 , or -P(J) 2 ;
  • Z is -COOH, -H, -S0 3 H, -P0 3 H 2 , -N(J) 2 , or -P(J) 2 ; and i is an integer from 0 to .
  • the present invention is further directed to a metal chelate comprising a metal and the metal chelating composition of the present invention.
  • the present invention is further directed to a process for the purification or detection of a polypeptide or other composition having an affinity for a metal chelate.
  • the process comprising contacting the composition with a metal chelate, the metal chelate comprising a metal and the metal chelating composition of the present invention.
  • the present invention is further directed to a process for the preparation of a mono- or dicarboxylated amine .
  • the process comprises combining an amine and an oxoacid in the presence of a reducing agent .
  • the amine has the formula R 2 R 3 NH wherein R 2 is hydrocarbyl or substituted hydrocarbyl and R 3 is hydrogen, hydrocarbyl or substituted hydrocarbyl.
  • the linkage between the chelator and the resin was found by us to be an important parameter for the selectivity of the resin for polyhistidine tagged proteins.
  • Conventional nitrilotriacetic acid resin has a positively charged amine linkage that acts as a binding site for any negatively charged molecule which may interfere with the binding of the protein to the coordination sites offered by the immobilized metal.
  • Oxygen, sulfur, selenium and amides have some affinity for metals which may provide enhanced chelation properties, binding the metal more firmly than traditional tetradentate chelators having positive amine linkages.
  • the use of a non-charged atoms between the nitrilo nitrogen and the carrier appears to reduce non-specific binding of proteins.
  • the chelating compositions of the present invention correspond to composition (1) shown in the structure below:
  • Q is a carrier
  • S 1 is a spacer
  • A is an ether, thioether, selenoether, or amide linkage; T is a bond or substituted or unsubstituted alkyl or alkenyl ;
  • X is -(CH 2 ) k CH 3 , - (CH 2 ) k C00H,- (CH 2 ) k S0 3 H, - (CH 2 ) k P0 3 H 2 , - (CH 2 ) k N(J) 2 , or - (CH 2 ) k P(J) 2 , preferably -(CH 2 ) k C00H or -(CH 2 ) k S0 3 H; k is an integer from 0 to 2;
  • J is hydrocarbyl or substituted hydrocarbyl
  • Y is -COOH, -H, -S0 3 H, -P0 3 H 2 , -N(J) 2 , or -P(J) 2 , preferably, -COOH;
  • Z is -COOH, -H, -S0 3 H, -P0 3 H 2 , -N(J) 2 , or -P(J) 2 , preferably, -COOH; and i is an integer from 0 to 4 , preferably 1 or 2.
  • the carrier, Q may comprise any solid or soluble material or compound capable of being derivatized for coupling.
  • Solid (or insoluble) carriers may be selected from a group including agarose, cellulose, methacrylate copolymers, polystyrene, polypropylene, paper, polyamide, polyacrylonitrile, polyvinylidene, polysulfone, nitrocellulose, polyester, polyethylene, silica, glass, latex, plastic, gold, iron oxide and polyacrylamide, but may be any insoluble or solid compound able to be derivatized to allow coupling of the remainder of the composition to the carrier, Q.
  • a preferred solid carrier is agarose or a high- throughput screening microtiterplate .
  • Soluble carriers include proteins, nucleic acids including DNA, RNA, and oligonucleotides, lipids, liposomes, synthetic soluble polymers, proteins, polyamino acids, albumin, antibodies, enzymes, streptavidin, peptides, hormones, chromogenic dyes, fluorescent dyes, flurochromes or any other detection molecule, drugs, small organic compounds, polysaccharides and any other soluble compound able to be derivatized for coupling the remainder of the composition to the carrier, Q. Proteins or polysaccharides are the preferred carrier.
  • the spacer, S 1 which flanks the carrier comprises a chain of atoms which may be saturated or unsaturated, substituted or unsubstituted, linear or cyclic, or straight or branched.
  • the chain of atoms defining the spacer, S 1 will consist of no more than about 25 atoms; stated another way, the backbone of the spacer will consist of no more than about 25 atoms. More preferably, the chain of atoms defining the spacer, S 1 , will consist of no more than about 15 atoms, and still more preferably no more than about 12 atoms.
  • the chain of atoms defining the spacer, S 1 will typically be selected from the group consisting of carbon, oxygen, nitrogen, sulfur, selenium, silicon and phosphorous and preferably from the group consisting of carbon, oxygen, nitrogen, sulfur and selenium.
  • the chain may optionally include one or more ether, thioether, selenoether, amide, or amine linkages between hydrocarbyl or substituted hydrocarbyl regions.
  • Exemplary spacers, S 1 include methylene, alkyleneoxy (-(CH 2 ) a O-), alkylenethioether (-(CH 2 ) a S-), alkyleneselenoether (- (CH 2 ) a Se-) , alkyleneamide
  • the spacer, S 1 is a hydrophilic, neutral structure and does not contain any amine linkages or substituents or other linkages or substituents which could become electrically charged during the purification of a polypeptide.
  • L is -A-T-CH(X)-
  • the chelating composition corresponds to the formula:
  • T is preferably substituted or unsubstituted C_ to C 6 alkyl or substituted or unsubstituted C 2 to C 6 alkenyl.
  • A is -S-
  • T is -(CH 2 ) n -
  • n is an integer from 0 to 6, typically 0 to 4 , and more typically 0, 1 or 2.
  • the chelating composition corresponds to the formula:
  • the sequence -S 1 -L- 7 in combination is a chain of no more than about 35 atoms selected from the group consisting of carbon, oxygen, sulfur, selenium, nitrogen, silicon and phosphorous, more preferably only carbon, oxygen sulfur and nitrogen, and still more preferably only carbon, oxygen and sulfur.
  • nitrogen when present, is preferably in the form of an amide moiety.
  • the carbon chain atoms are substituted with anything other than hydrogen, they are preferably substituted with hydroxy or keto.
  • L comprises a portion (sometimes referred to as a fragment or residue) derived from an amino acid such as cystine, homocystine, cysteine, homocysteine, aspartic acid, cysteic acid or an ester thereof such as the methyl or ethyl ester thereof .
  • exemplary chelating compositions of the present invention include the following:
  • metal chelating compositions (1) of the present invention may be derived from compositions having the general formula:
  • composition (2) wherein A, T, X, Y, Z and i are as previously defined.
  • composition (2) is represented by one of the following formulae:
  • Compositions corresponding to structure (2) in which at least one of X, Y and Z comprises a carboxylic acid moiety may be prepared by reductive alkylation of an amine.
  • mono- and dicarboxylated amines may be prepared by reacting an amine having the formula R 2 R 3 NH wherein R 2 is hydrocarbyl or substituted hydrocarbyl and R 3 is hydrogen, hydrocarbyl or substituted hydrocarbyl with an oxoacid, such as glyoxylic acid, in the presence of a reducing agent such as a pyridine-borane complex, dimethylborane , trimethylborane, sodium cyanoborohydride .
  • a nitrilotriacetic acid derivative of cystine may be prepared by combining cystine, an oxoacid such as glyoxylic acid, and a mild reducing agent; alcohol may preferably be included to aid in the clarification of the solution.
  • other methods known in the art may be used for the preparation of composition (2) , including haloalkylacids .
  • Composition (2) may be immobilized to form composition (1) by covalently attaching a chemical spacer, S 1 , to the linker, L, by any method known in the art and then reacting the carrier with the spacer, S 1 , to form a carrier-spacer chelate complex of composition (1).
  • carrier Q is first reacted with the spacer, S 1 , to form a carrier-spacer complex. Thereafter, the carrier-spacer complex is attached to the chelate-complex through the linker, L, to form composition (1) .
  • Q it is advantageous to activate the carrier, Q, with S 1 prior to the attachment of the chelating portion of the molecule.
  • Q is an agarose resin
  • it may be activated using epichlorohydrin, tetrabutyldiglycidyl ether or any substance capable of activating a carrier.
  • a metal chelate may be formed by addition of a metal or a metal oxide to chelating composition (1) or composition
  • a metal chelate of the present invention (in immobilized form) is represented by the following formula:
  • M comprises any metal or metal oxide capable of forming a chelate.
  • Preferred metals and metal oxides include Ni, Hg, Ga, Cu, Ru, Co, Cd, Mg, Mn, Ti , In, Zn, Tc, Rh, Pd, Re, Fe, Au, Pb, and Bi, with Fe, Cu, Co, Au, and Ni being preferred for most applications.
  • the metal, M preferred for a given application is dependant upon the specific binding capabilities of the chelating portion of composition (1) or (2) and on the compound to be bound or purified.
  • M is optimally Ni for purifying proteins with poly histidine sequences.
  • M is optimally Fe or Ga .
  • hydrocarbyl moieties described herein are organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, and aryl moieties. These moieties may be substituted or unsubstituted and preferably are substituted or unsubstituted alkyl. These moieties also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbyl groups, such as alkaryl, alkenaryl and alkynaryl . Unless otherwise specified, these moieties comprise 1 to 20 carbon atoms.
  • the alkyl groups described herein are preferably lower alkyl containing from one to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight, branched chain or cyclic and include methyl, ethyl, propyl , isopropyl, butyl, hexyl and the like. They may be substituted with aliphatic or cyclic hydrocarbyl radicals.
  • the alkenyl groups described herein are preferably lower alkenyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethenyl, propenyl , isopropenyl, butenyl, isobutenyl, hexenyl, and the like. They may be substituted with aliphatic or cyclic hydrocarbyl radicals.
  • the alkynyl groups described herein are preferably lower alkynyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethynyl, propynyl , butynyl , isobutynyl, hexynyl , and the like. They may be substituted with aliphatic or cyclic hydrocarbyl radicals. Unless otherwise specified, the aryl moieties described herein contain from 6 to 20 carbon atoms and include phenyl . They may be hydrocarbyl substituted with the various substituents defined herein. Phenyl is the more preferred aryl .
  • the substituted hydrocarbyl moieties described herein are hydrocarbyl moieties which are substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted with a hetero atom such as nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen atom.
  • substituents are other than hydroxyl and include lower alkoxy such as methoxy, ethoxy, butoxy; halogen such as chloro or fluoro; ethers; acetals; ketals; esters; heteroaryl such as furyl or thienyl; alkanoxy; acyl; acyloxy; nitro; amino; and amido.
  • acyl moieties described herein contain hydrocarbyl, substituted hydrocarbyl or heteroaryl moieties. They have the general formula -C(0)X wherein X may include hydrocarbyl, hydrocarbyloxy, hydrocarbylamino or hydrocarbylthio .
  • a protein includes antibodies, enzymes, hemoglobin, hormones, polypeptides and peptides; and may be an intact molecule, a fragment thereof, or a functional equivalent thereof; and may be genetically engineered.
  • An antibody includes both polyclonal and monoclonal antibodies; and may be an intact molecule, a fragment thereof; and may be genetically engineered.
  • N, N-bis-Carboxymethyl-L-cysteine Agarose A solution of 16.3 g of L-cysteine in 185 ml of 1 N sodium hydroxide was added to 48 ml of epichlorohydrin activated resin in an Erlenmeyer flask and mixed gently for 18 hours at 22 °C. The mixture was washed five times with 200 ml of water. The Kaiser test for free amine [Kaiser, E., et al, Anal. Biochem. , 34:595 (1970)] gave a deep blue color.
  • Nickel loading and capacity analysis The N,N- bis (carboxymethyl) -cysteine resin prepared as described above was first tested by determining the ability of the resin to chelate nickel. Approximately 2.5 ml of the resin was incubated with 10 ml of 10 mg/ml nickel sulfate over night with shaking at 50 rpm at 4°C. The resin was mixed well and placed into 1 x 10 cm column and the extra nickel solution was allowed to flow out of the resin. The unbound nickel sulfate was rinsed from the resin using 20 to 30 column volumes of deionized water. The remaining water was allowed to drain out of the resin. An equal volume of water was added to the resin, mixed well and then the slurry was removed from the column for storage at 4°C. The equivalent of one ml of packed bed resin was acid hydrolyzed and then analyzed by ICP for nickel content. This resin bound 5.2 ⁇ mole of nickel per ml of resin.
  • Protein binding and specificity testing The use test for specific protein binding of poly histidine containing proteins was done on a 0.5 x 7.6 cm column containing 1.5 ml of packed bed resin. The resin was equilibrated with 50 mM sodium phosphate, 0.5 M sodium chloride and 10 mM imidazole pH 8.0 (equilibration buffer). Five ml of a crude E. coli extract containing a bacterial alkaline phosphatase with a poly-histidine tag was loaded onto the column. This crude extract was made fresh from frozen E. coli cell paste using CelLytic-B (Sigma Chemical Company) . The column was washed with 10 column volumes (15 ml) of equilibration buffer to remove unbound proteins .
  • the bound material was eluted with 10 column volumes (15 ml) 50 mM sodium phosphate, 0.5 M sodium chloride and 320 mM imidazole pH 8.0. The peak fractions of the eluted material were pooled. The eluted material was assayed for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , alkaline phosphatase activity and protein content by Bradford protein assay. The assay results demonstrated that the eluted protein from this one step isolation procedure using this unique chelate resin gave essentially homogeneous bacterial alkaline phosphatase.
  • L-cystine 24.03 g; 0.1 moles
  • Glyoxylic acid monohydrate
  • the solution contained 20 ml buffer per mmole cystine and 40 mole glyoxylic acid per mole cystine.
  • An ice-water bath was placed around the beaker to cool the solution to between 15 to 25°C.
  • the pH of the mixture was adjusted to 9.0 with 5 N sodium hydroxide.
  • the ice-bath was removed and the beaker was left at room temperature. 8 M borane-pyridine complex (250 mL) was added, 20 moles per mole cystine. Ethanol to a final concentration of between 25 to 50% was added to clarify the solution. The solution was stirred at room temperature for two hours to complete the reaction. The reaction was monitored by HPLC until the reaction was complete. The reaction mixture was poured into a larger vessel and diluted with 3 volumes of water. Hydrochloric acid (10 N) was added slowly to adjust the pH to less than 1.0. The reaction mixture was stirred 30 to 60 minutes. The pH of the reaction mixture was then adjusted using 5 N sodium hydroxide to about 6.5.
  • N,N,N' ,N' -tetrakis (carboxymethyl) -L- cystine The crude N,N,N' ,N' -tetrakis (carboxymethyl) -L- cystine reaction mix was diluted with water to lower the conductivity to less than 5 milli mhos. The pH was adjusted to between 7.0 and 8.5 with 1 M sodium hydroxide. The mixture was then applied to a DEAE-Sephadex ® -HC03 (Pharmacia Biotech) column that had been equilibrated in deionized water using 10 ml resin per mmole cystine starting material. When the column charge was complete, it was washed with 4 column volumes of deionized water.
  • the column was then washed with 4 column volumes 0.1 M triethylammonium bicarbonate (TEAB) .
  • TEAB triethylammonium bicarbonate
  • the purified product was eluted from the column with 0.5 M TEAB.
  • the pooled material was dried in a Rotovap until a tacky solid was formed. It was then dissolved in a minimum volume of water. The material was now dried completely. The water re-suspension and drying steps were then repeated once more. The final solid was dissolved in 400 mL water.
  • the amino Sepharose ® 6B was prepared by suspending the epichlorohydrin activated resin in an equal volume (one resin volume) of 2 M ammonium hydroxide and gently mixed overnight at room temperature. The resin was washed with 3 volumes of 0.1 M sodium phosphate, pH 7. The resin was washed with 6 volumes of deionized water
  • Bromoacetylation of amino Sepharose ® 6B The washed amino Sepharose ® 6B resin was added to water to make a 50% resin slurry. The pH was adjusted to about 5.8 to 6.0 with I N HCl . The resin slurry was maitained at room temperature and the following reactions were conducted in reduced light conditions. 0.15 moles of bromoacetic acid in 0.2 M imidazole pH 5.8 was added to the slurry. Then 0.75 moles solid l-ethyl-3- (3-diamethylaminopropyl) carbodiimide (EDAC) was added while the resin was being mixed gently. This resin slurry was gently mixed at room temperature for 4 hours while maintaining pH 6.0.
  • EDAC solid l-ethyl-3- (3-diamethylaminopropyl) carbodiimide
  • the unreacted amines were blocked by acetylation. 6.5 ml acetic anhydride was added to the resin slurry followed by gently mixing for 30 min. The resin was then washed with 5 volumes of 0.1 M sodium phosphate, pH 7.5.
  • Bis (Carboxymethyl) cysteine coupling to bromoacetylated resin The following reactions were conducted in reduced light conditions. The washed bromoacetylated resin cake was suspended in 1 resin volume of 0.1 M sodium phosphate, pH 7.5 to make a 50% resin slurry. The slurry was bubbled with nitrogen for 10 minutes to deaerate. 15 mmoles of bis (carboxymethyl) cysteine prepared as described in Example 2 was added and the pH was adjusted to 7.5 with 1 N sodium hydroxide. It was then mixed at room temperature for at least 2 hours.
  • ⁇ - mercaptoethanol (14.3 M, 1.5 mL per liter resin) was added to the slurry and the incubation was continued for at least 30 minutes at room temperature to block unreacted bromoacetyl groups. The resin was then washed with 3 volumes phosphate buffered saline followed by 5 volumes of deionized water.
  • Nickel loading and capacity analysis The N,N- bis (carboxymethyl) -L-cysteine prepared as described above was tested by first determining the ability of the material to chelate nickel as described in Example 1. This resin bound 12.0 ⁇ mole of nickel per ml of resin.
  • Protein binding and specificity testing The use test for specific protein binding of poly histidine containing proteins by this resin was carried out as described in Example 1. The assay results demonstrated that the eluted protein from this one step isolation procedure using this unique chelate resin gave essentially homogeneous bacterial alkaline phosphatase.
  • Example 4 The use test for specific protein binding of poly histidine containing proteins by this resin was carried out as described in Example 1. The assay results demonstrated that the eluted protein from this one step isolation procedure using this unique chelate resin gave essentially homogeneous bacterial alkaline phosphatase.
  • L-cysteic acid methyl ester A mixture of 1 g of L-cysteic acid, 8 ml of 4 N hydrochloric acid in dioxane and 30 ml dry methanol was placed in a bottle. The bottle was capped and stored for approximately 96 hours at room temperature. Thin layer chromatography of the clear solution [Analtech silica gel plates, n-butanol : ethyl acetate: acetic acid: water (1:1:1:1), chlorine/potassium iodide-starch reagent (Stewart. J.M and Young.
  • Aminoethylamido-N,N-bis- (carboxymethyl) -L-cysteic acid preparation Triethylamine (10 ml) was added to a mixture of the 0.8 g of the above cysteic acid methyl ester in 12.5 ml of DMF in a bottle to get a clear solution. To this mixture 4.4 g of bromoacetic acid was added followed by approximately 15 ml of triethylamine until the pH was approximately 10. The solution became a solid mass. After a month at room temperature an additional 1.5 g of bromoacetic acid and 15 ml of DMF were added to the mixture. The pH of the mixture was adjusted to approximately 10 with triethylamine.
  • the product was eluted with a linear gradient with 1 L each of water and 0.1 N hydrochloric acid. The flow rate was 2 ml per minute. Fractions of 5 ml were collected. Fractions containing yellow product were assayed. Based on thin layer chromatography [Analtech silica gel plates, n-butanol: ethyl acetate : acetic acid: water (1:1:1:1), chlorine/potassium iodide-starch] the fractions containing the product were pooled and evaporated to dryness under vacuum to obtain 295 mg of light yellow foamy solid. Mass spec analysis gave the m/z of the major M +1 of 328.5.
  • Nickel loading and capacity analysis The aminoethylamido-N-bis- (carboxymethyl) -L-cysteic acid resin prepared as described above was tested as described in Example 1. This resin bound 15.6 ⁇ mole of nickel per ml of resin. Protein binding and specificity testing: The use test for specific protein binding of poly histidine containing proteins by this resin was carried out as described in Example 1. The assay results demonstrated that the eluted protein from this one step isolation procedure using this unique chelate resin gave essentially homogeneous bacterial alkaline phosphatase.
  • Bovine serum albumin (272 mg; BSA) at 10 mg/ml was dissolved in 0.1 M sodium phosphate, pH 7.2.
  • Bromoacetic acid NHS ester (57 mg; 0.24 mmole) in 0.5 ml dimethyl formamide (DMF) was added to the solution and incubated for 2 hours at room temperature with stirring.
  • the reaction mixture was desalted on a Sephadex ® G-50 (Pharmacia Biotech) column equilibrated and run in 0.1 M sodium phosphate, pH 7.2. The column was monitored by absorbance at 280 nm. Fractions were combined that contained absorbance at 280 nm. They were then gently bubbled with Argon for 3 minutes.
  • N,N- bis (carboxymethyl) cysteine 39 mg; 0.12 mmoles was dissolved in 0.2 ml of 0.1 M sodium phosphate, pH 8.0 (as prepared in Example 3) and then added to the BSA solution, which was then bubbled with argon. The reaction mixture was incubated overnight at 4 °C. The coupling efficiency was monitored by the 5, 5 ' -dithio-bis (2-nitrobenzoic acid (DTNB) reaction and was determined to be at least 95 %.
  • DTNB 2,5 ' -dithio-bis (2-nitrobenzoic acid
  • the mixture was desalted on a Sephadex ® G-50 (Pharmacia Biotech) column eluted with a buffer containing 10 mM 3-(N- morpholino)propanesulfonic acid (MOPS) and 0.15 M sodium chloride, pH 7.0.
  • the soluble BSA-chelate was charged with 0.01 M nickel sulfate in a buffer of 10 mM MOPS and 0.15 M sodium chloride, pH 7.0.
  • the final solution was desalted on a Sephadex ® G-50 (Pharmacia Biotech) column.
  • the desalted blue-colored BSA nickel chelate had absorbance peaks at 280 nm and 390 nm indicating that nickel was chelated to the conjugate.
  • a model fusion protein, bacterial alkaline phosphatase, containing an N-terminal poly histidine tag was incubated in the BSA nickel chelate microtiter wells at various concentrations in the MOPS buffer. The plate was washed three times with the MOPS buffer to remove unbound protein. The alkaline phosphatase fusion protein was detected by incubation of the microtiter wells with an alkaline phosphatase enzyme substrate. The assay demonstrated a significant amount of chelate specific binding of the poly histidine fusion protein to the albumin nickel chelate.

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Publication number Priority date Publication date Assignee Title
WO2003087047A1 (en) * 2002-04-11 2003-10-23 Peptron Co., Ltd. Metal chelate composition, macromolecule complex thereof, preparing method thereof and use
WO2003091689A2 (en) * 2002-03-28 2003-11-06 Rutgers, The State Of University Of New Jersey Bis-transition-metal-chelate-probes
GB2398782A (en) * 2003-02-28 2004-09-01 Amersham Biosciences Ab A method of generating metal chelating affinity ligands
WO2004087284A1 (en) * 2003-04-04 2004-10-14 Amersham Biosciences Ab Preparation of a metal chelating separation medium
WO2005029075A1 (en) * 2003-09-17 2005-03-31 Rutgers, The State University Of New Jersey Bis-transition-metal-chelate probes
EP1581337A2 (en) * 2002-10-18 2005-10-05 Promega Corporation Composition for separating molecules
EP1608765A2 (en) * 2003-02-24 2005-12-28 New England Biolabs, Inc. Overexpression, purification and characterization of a thermolabile phosphatase
EP1656443A2 (en) * 2003-05-02 2006-05-17 Sigma-Aldrich Co. Solid phase cell lysis and capture platform
JP2007524804A (ja) * 2003-01-22 2007-08-30 セレネックス, インコーポレイテッド アルキル連結ヌクレオチド組成物
US7282475B2 (en) 2004-08-25 2007-10-16 Sigma-Aldrich Co. Compositions and methods employing zwitterionic detergent combinations
WO2007137752A1 (en) * 2006-05-26 2007-12-06 Ge Healthcare Bio-Sciences Ab A method for generating metal chelating affinity ligands
EP2022561A1 (en) * 2007-08-06 2009-02-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Immobilisation of chelating groups for immobilised metal ion chromatography (IMAC)
US7741053B2 (en) 2005-05-13 2010-06-22 Sigma-Aldrich Co. Processes for purification of recombinant proteins
US9073971B2 (en) 2007-08-06 2015-07-07 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Immobilization of chelating groups for immobilized metal ion chromatography (IMAC)
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
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Families Citing this family (24)

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US20060141554A1 (en) * 2002-09-12 2006-06-29 Gee Kyle R Site-specific labeling of affinity tags in fusion proteins
US20050233307A1 (en) * 2002-09-12 2005-10-20 Kyle Gee Site-specific labeling of affinity tags in fusion proteins
GB0406015D0 (en) * 2004-03-17 2004-04-21 Dynal Biotech Asa Improvements in magnetic polymer particles
US7153574B2 (en) * 2004-07-16 2006-12-26 Xerox Corporation Surface grafted metal oxide particles and compositions comprising the same
JP4456952B2 (ja) 2004-07-16 2010-04-28 富士ゼロックス株式会社 電子写真感光体、プロセスカートリッジおよび電子写真装置
JP4456953B2 (ja) * 2004-07-16 2010-04-28 富士ゼロックス株式会社 画像形成装置及びプロセスカートリッジ
JP4456954B2 (ja) * 2004-07-16 2010-04-28 富士ゼロックス株式会社 電子写真感光体、プロセスカートリッジおよび電子写真装置
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HU230585B1 (hu) 2012-05-30 2017-01-30 Budapesti Műszaki és Gazdaságtudományi Egyetem Fehérjék megkötésére és elválasztására alkalmas lantanida fémionokkal komplexált hordozók
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DK3887033T3 (da) 2018-11-28 2024-08-12 Cube Biotech Gmbh Chelatdannende materiale i fast fase, metode til fremstilling heraf og anvendelse heraf til oprensning af proteiner
WO2024026446A1 (en) 2022-07-29 2024-02-01 Genentech, Inc. Method for purifying a polypeptide comprising two imac steps and apparatus therefore

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0189373A2 (de) * 1985-01-22 1986-07-30 Ciba-Geigy Ag Anionisch modifizierte Polysaccharide, Verfahren zu deren Herstellung und deren Verwendung
EP0253303A2 (de) * 1986-07-10 1988-01-20 F. Hoffmann-La Roche Ag Neue Metallchelatharze
WO1993011735A1 (en) * 1991-12-18 1993-06-24 Advanced Oxygen Technologies, Inc. Treatment of fungal infections

Family Cites Families (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2205995A (en) 1937-01-20 1940-06-25 Ig Farbenindustrie Ag Production of amino carboxylic acid nitriles
US2885428A (en) 1958-01-08 1959-05-05 Rohm & Haas 2-isothiocyanatoisocamphane
IT754322A (ja) 1965-01-25
US3959342A (en) 1970-08-24 1976-05-25 Bethlehem Steel Corporation Process for the preparation of nitrilotriacetonitrile (NTN)
JPS512708A (ja) * 1974-06-28 1976-01-10 Lion Fat Oil Co Ltd Senjozaisoseibutsu
JPS55139347A (en) 1979-04-12 1980-10-31 Nippon Paint Co Ltd Amino compound
SE449225B (sv) 1980-12-18 1987-04-13 Gelinnovation Hb Gelprodukt baserad pa partiklar innehallande galaktan for separationsendamal, molekyl- och partikelimmobilisering
SE451802B (sv) * 1984-05-10 1987-11-02 Jerker Porath Produkt for adsorption av metalljoner
SE470099B (sv) 1984-05-17 1993-11-08 Jerker Porath Sulfonaktiverade tioeteradsorbenter för separation av t ex protein
SE454885B (sv) 1984-10-19 1988-06-06 Exploaterings Ab Tbf Polymerbelagda partiklar med immobiliserade metalljoner pa sin yta jemte forfarande for framstellning derav
SE452557B (sv) 1984-10-30 1987-12-07 Exploaterings Ab Tbf Amfipatisk gelprodukt for kromatografisk och satsvis adsorption
US4569794A (en) 1984-12-05 1986-02-11 Eli Lilly And Company Process for purifying proteins and compounds useful in such process
US5047513A (en) * 1986-07-10 1991-09-10 Hoffmann-La Roche Inc. Metal chelate resins
CA1328537C (en) 1986-07-10 1994-04-12 Heinz Dobeli Metal chelate resins
CA1340522C (en) 1987-03-10 1999-05-04 Heinz Dobeli Fusion proteins containing neighbouring histidines for improved purification
US5061641A (en) 1988-04-01 1991-10-29 Immunomedics, Inc. Method for radiolabeling proteins
US5075099A (en) 1988-05-31 1991-12-24 Neorx Corporation Metal radionuclide chelating compounds for improved chelation kinetics
US5287933A (en) * 1991-10-22 1994-02-22 Caterpillar Pavin, Products Inc. Hood support assembly for an earth working machine
JP3374442B2 (ja) 1993-06-02 2003-02-04 株式会社豊田自動織機 自動車の外気導入装置
JP3704172B2 (ja) 1993-10-25 2005-10-05 日本メジフィジックス株式会社 キレート形成性フェニルジアミノジチオール誘導体及 びその用途
CA2111287C (en) 1993-12-13 2007-07-03 Graham Darling Chelating resins and use thereof in selectively recovering valuable metals from acid mine drainage
WO1995021179A1 (de) 1994-02-07 1995-08-10 Qiagen Gmbh Verfahren zur abreicherung oder entfernung von endotoxinen
US5990301A (en) 1994-02-07 1999-11-23 Qiagen Gmbh Process for the separation and purification of nucleic acids from biological sources
DE4430023A1 (de) 1994-08-24 1996-02-29 Boehringer Mannheim Gmbh Elektrochemischer Sensor
US5789578A (en) 1996-01-11 1998-08-04 Massey University Methods for the preparation of resins with ligands attached thereto through a linking group comprising sulfide, sulfoxide or sulfone functionality
EP0958055B1 (en) 1996-06-24 2005-02-09 Ibc Advanced Technologies, Inc. Poly n-cyclic aromatic ligands bonded to solid supports for removing and concentrating ions from solutions
JPH10182570A (ja) 1996-12-27 1998-07-07 Fuji Photo Film Co Ltd アミノポリカルボン酸系キレート剤、その重金属化合物、写真用添加物、および処理方法
US6004529A (en) 1997-04-11 1999-12-21 Nycomed Imaging As Chelating agents
US6623655B1 (en) 2000-04-24 2003-09-23 Sigma-Aldrich Co. Metal chelating compositions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0189373A2 (de) * 1985-01-22 1986-07-30 Ciba-Geigy Ag Anionisch modifizierte Polysaccharide, Verfahren zu deren Herstellung und deren Verwendung
EP0253303A2 (de) * 1986-07-10 1988-01-20 F. Hoffmann-La Roche Ag Neue Metallchelatharze
WO1993011735A1 (en) * 1991-12-18 1993-06-24 Advanced Oxygen Technologies, Inc. Treatment of fungal infections

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANSPACH F B: "SILICA-BASED METAL CHELATE AFFINITY SORBENTS I. PREPARATION AND CHARACTERIZATION OF IMINODIACETIC ACID AFFINITY SORBENTS PREPARED VIA DIFFERENT IMMOBILIZATION TECHNIQUES" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 672, no. 1/2, 1994, pages 35-49, XP000996948 ISSN: 0021-9673 *
PORATH J ET AL: "IMMOBILIZED METAL ION AFFINITY ADSORPTION AND IMMOBILIZED METAL IONAFFINITY CHROMATOGRAPHY OF BIOMATERIALS. SERUM PROTEIN AFFINITIES FOR GEL-IMMOBILIZED IRON AND NICKEL IONS" BIOCHEMISTRY,US,AMERICAN CHEMICAL SOCIETY. EASTON, PA, vol. 22, 1983, pages 1621-1629, XP002060578 ISSN: 0006-2960 cited in the application *
PORATH J ET AL: "METAL CHELATE AFFINITY CHROMATOGRAPHY, A NEW APPROACH TO PROTEIN FRACTIONATION" NATURE,GB,MACMILLAN JOURNALS LTD. LONDON, vol. 258, 18 December 1975 (1975-12-18), pages 598-599, XP002059246 ISSN: 0028-0836 cited in the application *
See also references of EP1276716A2 *

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WO2003091689A2 (en) * 2002-03-28 2003-11-06 Rutgers, The State Of University Of New Jersey Bis-transition-metal-chelate-probes
WO2003091689A3 (en) * 2002-03-28 2004-12-23 Rutgers The State Of Universit Bis-transition-metal-chelate-probes
WO2003087047A1 (en) * 2002-04-11 2003-10-23 Peptron Co., Ltd. Metal chelate composition, macromolecule complex thereof, preparing method thereof and use
JP2006502857A (ja) * 2002-10-18 2006-01-26 プロメガ コーポレイション 分子を分離するための方法
EP1581337A4 (en) * 2002-10-18 2008-05-07 Promega Corp COMPOSITION FOR DISCONNECTING MOLECULES
EP1585970A4 (en) * 2002-10-18 2008-08-06 Promega Corp METHOD FOR SEPARATING MOLEK LEN
EP1581337A2 (en) * 2002-10-18 2005-10-05 Promega Corporation Composition for separating molecules
EP1585970A2 (en) * 2002-10-18 2005-10-19 Promega Corporation Methods for separating molecules
JP2007524804A (ja) * 2003-01-22 2007-08-30 セレネックス, インコーポレイテッド アルキル連結ヌクレオチド組成物
EP1608765A2 (en) * 2003-02-24 2005-12-28 New England Biolabs, Inc. Overexpression, purification and characterization of a thermolabile phosphatase
US7319014B2 (en) 2003-02-24 2008-01-15 New England Biolabs, Inc. Overexpression, purification and characterization of a thermolabile phosphatase
EP1608765A4 (en) * 2003-02-24 2006-10-18 New England Biolabs Inc OVEREXPRESSION, CLEANING AND CHARACTERIZATION OF A THERMOLABILENE PHOSPHATASE
GB2398782B (en) * 2003-02-28 2005-10-19 Amersham Biosciences Ab A method of generating metal chelating affinity ligands
US7005071B2 (en) 2003-02-28 2006-02-28 Amersham Biosciences Ab Method of generating metal chelating affinity ligands
KR101060896B1 (ko) * 2003-02-28 2011-08-30 지이 헬스케어 바이오-사이언시스 에이비 금속 킬레이트 친화성 리간드의 형성 방법
WO2004076475A1 (en) * 2003-02-28 2004-09-10 Amersham Biosciences Ab A method of generating metal chelating affinity ligands
GB2398782A (en) * 2003-02-28 2004-09-01 Amersham Biosciences Ab A method of generating metal chelating affinity ligands
JP2007528835A (ja) * 2003-02-28 2007-10-18 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 金属キレートアフィニティリガンドの合成方法
US7175767B2 (en) 2003-04-04 2007-02-13 Ge Healthcare Bio-Sciences Ab Preparation of a metal chelating separation medium
WO2004087284A1 (en) * 2003-04-04 2004-10-14 Amersham Biosciences Ab Preparation of a metal chelating separation medium
EP1656443A4 (en) * 2003-05-02 2007-06-06 Sigma Aldrich Co SOLID PHASE CELLYSIS AND IMPACT PLATFORM
EP1656443A2 (en) * 2003-05-02 2006-05-17 Sigma-Aldrich Co. Solid phase cell lysis and capture platform
WO2005029075A1 (en) * 2003-09-17 2005-03-31 Rutgers, The State University Of New Jersey Bis-transition-metal-chelate probes
US7282475B2 (en) 2004-08-25 2007-10-16 Sigma-Aldrich Co. Compositions and methods employing zwitterionic detergent combinations
US7897376B2 (en) 2004-08-25 2011-03-01 Sigma-Aldrich Co. Method for extracting a target product from a host cell employing zwitterionic detergent combinations
US7741053B2 (en) 2005-05-13 2010-06-22 Sigma-Aldrich Co. Processes for purification of recombinant proteins
WO2007137752A1 (en) * 2006-05-26 2007-12-06 Ge Healthcare Bio-Sciences Ab A method for generating metal chelating affinity ligands
US7988858B2 (en) 2006-05-26 2011-08-02 Ge Healthcare Bio-Sciences Ab Method for generating metal chelating affinity ligands
EP2022561A1 (en) * 2007-08-06 2009-02-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Immobilisation of chelating groups for immobilised metal ion chromatography (IMAC)
US9073971B2 (en) 2007-08-06 2015-07-07 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Immobilization of chelating groups for immobilized metal ion chromatography (IMAC)
EP2826555A3 (en) * 2007-08-06 2016-01-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Immobilisation of chelating groups for immobilised metal ion chromatography
US9675960B2 (en) 2007-08-06 2017-06-13 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Immobilization of chelating groups for immobilized metal ion chromatography (IMAC)
US10538552B2 (en) 2007-08-06 2020-01-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Immobilisation of chelating groups for immobilised metal ion chromatography (IMAC)
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
US9347027B2 (en) 2012-12-19 2016-05-24 Rohm And Haas Company Automatic dishwashing detergent

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DE60111628D1 (de) 2005-07-28
EP1276716B1 (en) 2005-06-22
ES2244609T3 (es) 2005-12-16
AU2001251479A1 (en) 2001-11-07
EP1627683A3 (en) 2006-12-20
WO2001081365A3 (en) 2002-03-28
US7709612B2 (en) 2010-05-04
US20060148090A1 (en) 2006-07-06
DE60111628T3 (de) 2009-04-02
US7033520B2 (en) 2006-04-25
US6623655B1 (en) 2003-09-23
ES2244609T5 (es) 2009-03-16
ATE298321T1 (de) 2005-07-15
EP1627683A2 (en) 2006-02-22
JP2003531213A (ja) 2003-10-21
DE60111628T2 (de) 2006-05-11
JP4298201B2 (ja) 2009-07-15
EP1276716A2 (en) 2003-01-22
US20040092031A1 (en) 2004-05-13

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